International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2207–2215 DOI 10.1099/ijs.0.047894-0

Cauliform lacking phospholipids from an abyssal hydrothermal vent: proposal of Glycocaulis abyssi gen. nov., sp. nov., belonging to the family Hyphomonadaceae

Wolf-Rainer Abraham,1 Heinrich Lu¨nsdorf,1 Marc Vancanneyt2 and John Smit3

Correspondence 1Helmholtz Center for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany Wolf-Rainer Abraham 2Laboratorium voor Microbiologie, Universiteit Gent, Belgium wolf-rainer.abraham@helmholtz- 3 hzi.de Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada

Cauliform bacteria are prosthecate bacteria often specialized for oligotrophic environments. A polyphasic approach, comprising 16S rRNA gene sequencing, lipid analysis and salt tolerance characterizations, was used to clarify the of one isolate, strain MCS 33T, obtained from above the hot water plume of a deep-sea hydrothermal vent near Vancouver island, Canada. Cells contained no detectable phospholipids or sulpholipids, but did contain 1,2-di-O-acyl-3-O-a-D- glucopyranosylglycerol, 1,2-di-O-acyl-3-O-a-D-glucopyranuronosylglycerol and the novel lipid 1,2-di-O-acyl-3-[O-a-D-glucopyranuronosyl]glycerol-69-N-glycine. It is assumed that the various glucoronosyl lipids are replacing, at least partially, the phospholipids in their various tasks in the cell cycle. The G+C content of the genomic DNA of strain MCS 33T was 62.8 mol%, and Q10 was the predominant respiratory ubiquinone. The 16S rRNA gene sequence of this chemoheterotrophic, aerobic, moderately halophilic strain showed only a low similarity of 94.4 % to that of Oceanicaulis alexandrii C116-18T, and both strains also differed based on their lipids. Although the novel strain was isolated from seawater sampled near a hydrothermal vent, its

optimum temperature for growth was 30 6C. The main cellular fatty acids were C18 : 1v7c,C18 : 0

and the unknown fatty acid ECL 11.798, and the main hydroxy fatty acid was C12 : 0 3-OH. The strain is proposed to represent a novel of a new , Glycocaulis abyssi gen. nov., sp. nov. The type strain of the type species is MCS 33T (5LMG 27140T5CCUG 62981T).

For decades bacteria having a stalk and reproducing with this is that these bacteria exhibit the physiological regularly by the separation of two cells that are morpho- properties of oligotrophs (Poindexter, 1981). Henrici and logically and behaviourally different from each other were Johnson (1935) placed bacteria possessing these character- regarded as members of the genus Caulobacter (Poindexter istics into the new genus Caulobacter. Stahl et al. (1992) 1964). One descendant is non-motile, sessile due to analysed the phylogeny of a number of caulobacteria and adhesion to the substratum and prosthecate, possessing a found low levels of similarity between sequences of the 16S tubular appendage of variable length – a prostheca (Staley rRNA gene. We analysed a large number of strains for their 1968). The other descendant is actively motile by means of pattern of proteins, polar lipids and 16S rRNA gene one polar flagellum. The mode of reproduction of the sequences (Abraham et al., 1997) and revealed the dimorphic prosthecate bacteria is regarded as a reflection paraphyletic nature of marine caulobacteria belonging of an ecological adaptation helping to disperse the mainly to the two genera Brevundimonas (Segers et al., population at each generation and thereby minimizing 1994) and Maricaulis (Abraham et al., 1999). The marine competition between descendants for resources. Consistent isolates show particularly high diversity, with strains differing so much from the genus Maricaulis that they Abbreviation: FAME, fatty acid methyl ester. cannot be included in this genus but belong to separate The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene genera, i.e. Oceanicaulis (Stro¨mpl et al., 2003) and sequence of strain MCS 33T is AJ227811. Woodsholea (Abraham et al., 2004). We describe here T Three supplementary figures are available with the online version of this another of these isolates, MCS 33 , which does not fit in paper. any of the described genera.

047894 G 2013 IUMS Printed in Great Britain 2207 W.-R. Abraham and others

The strains used in this study were obtained from the American type Culture Collection (ATCC), the Deutsche Sammlung fu¨r Mikroorganismen und Zellkulturen (DSMZ), the Department of Microbiology and Immunology at the University of British Columbia (MCS strains, Maricaulis virginensis VC-5T, Woodsholea maritima CM243T) and from C. Stro¨mpl, Helmholtz Center for Infection Research, Braunschweig, Germany (Oceanicaulis alexandrii C116- 18T). The strains were grown in the marine medium SPYEM: 30 g sea salts (Sigma), 0.5 g NH4Cl, 1 litre MilliQ-water. After autoclaving and cooling to ambient temperature, 20 ml 506PYE, 2 ml 50 % glucose (sterile) and 5 ml riboflavin (0.2 mg ml21) sterile filtered were added. 506PYE includes 100 g peptone and 50 g yeast extract in 1 litre deionized water (autoclaved). The strains were grown in 2-litre Erlenmeyer flasks at 30 uC and 100 r.p.m. and the biomass was harvested in the late exponential phase after 72 h. For electron microscopy, growing cells were prepared for negative staining, embedding and ultrathin sectioning as described in detail by Yakimov et al. (1998). Cells of strain MCS 33T showed morphological features typical of caulobacteria, when analysed by transmission electron microscopy. During cell division two morphotypes of daughter cells were observed: first, the swarmer cell, which is monopolarly and monotrichously flagellated; and, T Fig. 1. Ultrastructure of cells of strain MCS 33 . (a) Negatively second, the prosthecate daughter cell with its stalk stained dividing cells, which show characteristic caulobacterial (diameter 85 nm), ending in a spherical holdfast (diameter T features. One daughter cell is the swarmer cell and carries a single 140 nm) (Fig. 1a). Typically, strain MCS 33 formed flagellum. It is still connected to the second prosthecate daughter groups or clusters of cells (Fig. 1b), often bundled by cell by an extended septum. The stalk is terminated by a aggregated holdfasts (Fig. 1c, circle). Overall cell shape was characteristic spherical holdfast. (b) Survey view of a cluster of vibrioid and cell length ranged from 1.3 to 2.5 mm and cell cells, which show prostheca of different developmental states. (c) width from 0.65 to 0.72 mm. The interior part of the stalk Ultrathin section. A longitudinal cut shows the vibrioid shape of the appeared only slightly electron-dense and was surrounded cell body, which contains a pronounced chromosome, surrounded by the electron-translucent periplasm. The cell-wall by densely packed cytoplasm. Many prostheca end up in a tight architecture was Gram-negative; an outer membrane, cluster of holdfasts and form a typical rosette-like ensemble (white which was intensely undulated, formed the outer boundary circle). (d) Detailed view of the cell wall, showing the outer of the cell (Fig. 1c, d). membrane and cytoplasmic membrane. The periplasm appears as an electron-translucent matrix. fl, Flagellum; es, extended septum; Genomic DNA was isolated from two loopfuls of bacterial pr, prosthecum; hf, holdfast; cp, cytoplasm; om, outer membrane; cells using the DNeasy Blood and Tissue kit for purification cm, cytoplasmic membrane; stars, chromosome. Bars 1 mm (a, b), of total DNA (Qiagen) with the addition of RNase A 500 nm (c), 50 nm (d). (Sigma), according to the manufacturers’ instructions. DNA was enzymically digested as described by Gehrke et al. (1984) and the mean G+C content was determined by bootstrap consensus tree using the neighbour-joining HPLC (Tamaoka & Komagata, 1984). Calculations were algorithm (Fig. 2), maximum-parsimony (Fig. S1, available carried out according to Mesbah et al., (1989), with non- in IJSEM Online) and maximum linkage (Fig. S2) with methylated lambda-phage DNA (Sigma) as a standard. MEGA version 5.0.5 (Tamura et al., 2011), based on 1000 Amplification of the 16S rRNA gene and sequencing was resamplings and with Caulobacter vibrioides VKM1496T as done as described previously (Abraham et al., 2010). The an outgroup. phylogenetic position of strain MCS 33T was determined by analysis of the 16S rRNA gene sequence (Abraham et al., Isoprenoid quinones were extracted with chloroform/ 1999) using the software CLUSTAL W (Thompson et al., methanol (2 : 1, v/v) and analysed by the method published 1997). The 16S rRNA gene sequence showed 94.2 % by Minnikin et al. (1984). The main ubiquinone of strain similarity to that of both Maricaulis maris ATCC 15268T MCS33T was ubiquinone Q-10. For whole-cell fatty acid and O. alexandrii C116-18T and 93.5 % to W. maritima analysis, cells were saponified [15 % (w/v) NaOH, 30 min, CM243T. Alignment of sequences from the EMBL database 100 uC], methylated to fatty acid methyl esters (FAMEs) (Karsch-Mizrachi et al., 2012) was used to construct a (methanolic HCl, 10 min, 80 uC) and extracted [hexane/

2208 International Journal of Systematic and Evolutionary Microbiology 63 Glycocaulis abyssi gen. nov., sp. nov.

T rosenbergii VP6 (AF082795)

T 89 Hyphomonas hirschiana VP5 (AF082794)

100 T 0.01 Hyphomonas neptunium LE670 (AF082798)

T 51 Hyphomonas polymorpha DSM 2665 (AJ227813)

T Hyphomonas adhaerens MHS-3 (AF082790) 100 T 100 Hyphomonas jannaschiana VP2 (AJ227814)

T Hyphomonas oceanitis SCH89 (AF082797) 97 T 92 Hyphomonas johnsonii MHS-2 (AF082791)

T Ponticaulis koreensis GSW-23 (FM292497)

T litoralis SD10 (FJ230835) 83 92 T 100 Henriciella marina Iso4 (EF660760)

T 99 Henriciella aquimarina LMG 24711 (EU819081)

T 95 baltica IFAM 1418 (X52909)

T 100 Hirschia maritima GSW-2 (FM202386)

T Robiginitomaculum antarcticum IMCC3195 (EF495229)

T Hellea balneolensis DSM 19091 (AY576758) 100 T 94 Litorimonas taeanensis G5 (FJ230838)

T 99 Algimonas porphyrae 0C-2-2 (AB689189)

T 100 Oceanicaulis alexandrii C116-18 (AJ309863)

T 95 Oceanicaulis stylophorae GISW-4 (HM035090) 95 Glycocaulis abyssi MCS 33T (AJ227811)

T Woodsholea maritima CM243 (AJ578476)

T Maricaulis washingtonensis MCS 06 (AJ227804) 96 100 T Maricaulis salignorans MCS 18 (AJ227806)

97 Maricaulis maris ATCC 15268T (AJ227802)

T 86 Maricaulis virginensis VC-5 (AJ301667)

T 100 Maricaulis parjimensis MCS 25 (AJ227808)

Fig. 2. Neighbour-joining tree constructed with 16S rRNA gene sequences, showing the phylogenetic position of strain MCS 33T within the family Hyphomonadaceae. Evolutionary distances were computed using the method of Jukes & Cantor (1969). The sequence of Caulobacter vibrioides VKM 1496T (GenBank accession no. AJ227754; not shown) was used as an outgroup. Bootstrap values (.50 %) based on 1000 resamplings are shown at branch nodes. Bar, 0.01 substitutions per nucleotide position.

] methyl-tert-butyl ether (1 : 1, v/v) as described by but C12 : 0 3-OH and C12 : 1 3-OH were present while sopceis Osterhout et al. (1991). FAMEs were analysed on a of the genera Maricaulis were the only ones in the Hewlett Packard (HP) 5890A gas chromatograph. Hyphomonadaceae where iso-C11 : 0 3-OH, iso-C17 : 0, iso- Separation of FAMEs was achieved with a fused-silica C17 : 1v9c and C18 : 1v9c were present but C12 : 0 3-OH and capillary column (25 m by 0.2 mm) with cross-linked 5 % C12 : 1 3-OH were absent (Abraham et al., 1999). An phenyl methyl silicone (film thickness 0.33 mm; HP Ultra unknown fatty acid with ECL 11.798, which was absent in 2). The computer-controlled parameters were the same as members of the genera Maricaulis, Woodsholea and those described by Osterhout et al. (1991). The instrument Oceanicaulis, was found in considerable amounts in strain was equipped with a flame-ionization detector and an MCS 33T. The main hydroxy fatty acid of strain MCS 33T autosampler (HP 7673). H2 served as carrier gas. The was C12 : 0 3-OH, which was also found in species of the cellular fatty acid compositions of strain MCS 33T and the genera Oceanicaulis and Woodsholea but absent in members type strains of 10 recognized species of Hyphomonadaceae of the genus Maricaulis. In addition to C12 : 0 3-OH, strain T T are shown in Table 1. In strain MCS 33 , iso-C11 : 0 3-OH, MCS 33 contained also small amounts of C12 : 1 3-OH, iso-C17 : 0, iso-C17 : 1v9c,C18 : 1v9c and C15 : 0 were all absent which was not found in species of the genera Maricaulis, http://ijs.sgmjournals.org 2209 W.-R. Abraham and others

Table 1. Fatty acid content (mean % of total) of whole-cell hydrolysates of strain MCS 33T in comparison with members of the genera Maricaulis, Woodsholea, Oceanicaulis and Hyphomonas

Strains: 1, MCS 33T;2,M. maris ATCC 15268T;3,M. washingtonensis MCS 6T;4,M. salignorans MCS 18T;5,M. parjimensis MCS 25T;6,M. virginensis VC-5T;7,W. maritima CM243T;8,O. alexandrii C116-18T;9,O. stylophorae GISW-4T (data from Chen et al., 2012); 10, H. polymorpha DSM 2665T; 11, H. jannaschiana ATCC 33833T. Only fatty acids amounting to more than 1.0 % (mean) are shown. The following strains also T contained significant amounts (.1.0 %) of additional fatty acids: H. polymorpha DSM 2665 also contained summed feature 9 (C19 : 0 cyclo v10c, T T ECL 18.846 and/or ECL 18.858; 4.5 %); and O. alexandrii C116-18 contained 4.6 % and O. stylophorae GISW-4 1.1 % C19 : 0. tr, Trace amount (,1.0 %); 2, not detected.

Fatty acid 1 2 3 4 5 6 7 8 9 10 11

iso-C11 : 0 3-OH 2 2.6 tr tr tr 5.4 2222 ECL 11.798* 7.4 2222222222

C12 : 0 3-OH 3.0 222223.5 1.3 3.0 tr tr C12 : 1 3-OH tr 222222221.2 1.5 C15 : 0 2 tr tr tr 222221.9 1.7 ECL 15.275 2222226.1 2222

C16 : 0 4.6 17.0 11.2 8.9 3.6 9.8 1.4 2.2 2.3 1.9 10.4 Summed feature 4D 2 6.6 3.0 2.6 2.2 2.4 2222tr

C16 : 1v9c 2 1.0 tr tr 2222222 C17 : 0 2.9 5.3 9 8.7 7 15.3 2.2 18.0 6.9 18.3 9.7 C17 : 1v6c 1.8 tr 1.0 1.1 1.8 1.3 tr 3.9 2 15.3 4.2 C17 : 1v8c 2 4.0 10.2 10.0 4.7 9.6 2 1.0 2 10.9 4.8 iso-C17 : 0 2 7.7 9.6 10.8 1.7 6.9 22222 iso-C17 : 1v9c 2 17.4 22.4 28.0 3.9 13.8 22222 C18 : 0 10.8 1.1 tr tr 7.9 4.2 16.9 18.2 29.3 tr 3.7 C18 : 1v7c 68.7 24.5 16.2 12.9 47.9 13.0 65.4 29.1 27.8 21.7 48.4 C18 : 1v9c 2 6.4 10.7 7.7 6.0 3.4 22222 11 Methyl C18 : 1v5 tr 1.6 tr 1.0 tr 2.9 2 18.7 25.8 1.1 7.0 ECL 18.424 2 1.3 1.3 1.8 2.3 5.7 22222 ECL 18.797 2 tr tr tr 4.9 2.6 22220.3 5.0

*Unidentified fatty acid with equivalent chain-length of 11.798.

DSummed feature 4 comprised C18 : 1v7c/C18 : 1v9t/C18 : 1v12t/C18 : 1v7t (ECL 17.798).

Oceanicaulis or Woodsholea but was the main hydroxylated fatty acids, octadecenoic acid and octadecanoic acid, as fatty acid of species of the genus Hyphomonas. ions corresponding to the neutral loss of free fatty acids (m/z 572 and 570) and the corresponding ketenes (m/z 590 Polar lipids were extracted using a modified Bligh-Dyer and 588) were observed (Fig. S2). Furthermore, there were procedure (Bligh & Dyer 1959) as described previously a number of fragments in the upper mass region at (Vancanneyt et al. 1996) and analysed by fast atom intervals of about 14 mass units. These correspond to bombardment MS (Abraham et al. 1997). The polar lipids T fragmentations along the fatty acid acyl chains and of strain MCS 33 consisted entirely of glycolipids. No represent charge remote fragmentation similar to that phospholipids or sulpholipids were detected (Fig. S3). The observed in other polar lipids (Jensen et al. 1987). After the glycolipids were a-D-glucopyranosyl- and a-D-glucopyra- neutral losses of one fatty acid and the ketene of the other nuronosyl-diacylglycerols, also common in members of the one from the molecular ion at m/z 854, an ion of m/z genera Caulobacter, Brevundimonas, Maricaulis and some 306 Da was formed. This diagnostically important daugh- other . Using MS/MS, the structures of ter ion at m/z 306 fragmented further by the neutral loss of 21 of these glycolipids could be identified as listed in Table dehydro-glycerol, giving the even-numbered and hence N- 2. Additionally to these glycolipids, fast atom bombard- containing ion at m/z 232. A similar series of fragmentation ment MS revealed two ions with higher masses. Their leads via the neutral loss of the two fatty acids as ketenes to masses were even, requiring odd-numbered molecular the ion m/z 324, which loses dehydro-glycerol to the masses, suggesting the presence of nitrogen in the pyranosyl ion at m/z 250. The other minor component at molecules. To elucidate the structure of these lipids m/z 852 in the lipid fraction behaved in a similar way. The intensive MS studies were performed. The fast atom polar lipid therefore seemed to be a glucuronosyl-glycerol, bombardment collision-induced dissociation mass spec- which is esterified at the 6-position to glycine. The trum of the ion at m/z 854 showed the loss of two different observed daughter ions are in agreement with the

2210 International Journal of Systematic and Evolutionary Microbiology 63 Glycocaulis abyssi gen. nov., sp. nov.

Table 2. Polar lipids identified in strain MCS 33T by fast atom sodium chloride weak growth of strain MCS 33T was bombardment ionization and collision-induced dissociation MS observed and a salt concentration of 100 g l21 also allowed only weak growth. Good growth occurred between 20 and MGD, 1,2-di-O-acyl-3-O-a-D-glucopyranosylglycerol; MGDOx, 1,2- 60 g NaCl l21. By comparison, M. maris ATCC 15268T di-O-acyl-3-O-a-D-glucopyranuronosylglycerol; GGG, 1,2-di-O-acyl- showed a similar behaviour and at least moderate growth 3-[O-a-D-glucuronopyranosyl]glycerol-69-N-glycine. with 80–100 g NaCl l21. Temperature below 15 uCand above 40 uC inhibited growth of strain MCS 33T, which had Mass Type Fatty acids its growth optimum at 30 uC. M. maris could not grow below sn-1 sn-2 15 uCorat50uC; it showed its growth optimum at 40 uC. 726 MGD 16 : 1 – 16 : 1 728 MGD 16 : 1 – 16 : 0 Enzyme activity tests were conducted with API ZYM test 768 MGD 18 : 1 – 17 : 1 strips (bioMe´rieux), used according to the manufacturer’s 768 MGD 19 : 1 – 16 : 1 instructions. Substrate specificity tests were conducted with 770 MGD 18 : 1 – 17 : 0 API 20NE test strips (bioMe´rieux) and BIO Typ 100 770 MGDOx 18 : 1 – 16 : 0 (bioMe´rieux), according to the manufacturer’s instruc- 772 MGDOx 18 : 0 – 16 : 0 tions, at 30 uC for 4 days. A test was considered positive if a 784 MGDOx 18 : 1 – 17 : 0 colour change was noticeable. The results are given in the 784 MGDOx 18 : 0 – 17 : 1 species description. The characters that discriminate 796 MGDOx 18 : 1 – 18 : 1 between strain MCS 33T and its closest phylogenetic 798 MGDOx 18 : 1 – 18 : 0 neighbours are summarized in Table 2. 798 MGDOx 19 : 1 – 17 : 0 T 810 MGDOx 19 : 1 – 18 : 1 The entire lack of phospholipids in strain MCS 33 810 MGDOx 19 : 2 – 18 : 0 warrants discussion. Minnikin et al. (1974) reported on a 810 MGDOx 20 : 2 – 17 : 0 reduction of the phospholipid content of Brevundimonas 812 MGDOx 19 : 1 – 18 : 0 diminuta under phosphate limitation, and high levels of 822 MGDOx 20 : 1 – 18 : 2 glycolipids and sulpholipids were reported from marine 822 MGDOx 20 : 2 – 18 : 1 caulobacter strains, including M. maris (De Siervo, 1985), 822 MGDOx 20 : 3 – 18 : 0 reaching amounts as low as 0.3 % of the total lipids. We 824 MGDOx 20 : 1 – 18 : 1 also found considerable amounts of sulpholipids in 824 MGDOx 20 : 2 – 18 : 0 Maricaulis spp. but they were not detected in strain MCS 853 GGG 18 : 1 – 18 : 1 33T. Taking the various functions of phospholipids 855 GGG 18 : 1 – 18 : 0 identified in the cell cycle into account (Dowhan, 1997) it is puzzling how these functions can be replaced by glycolipids in strain MCS 33T. One possibility is that the postulated partial structure as indicated in the fragmenta- glycine lipids can replace some of the phospholipids. A tion scheme. The relative abundance of the carboxylate deeper understanding of the function of these glycolipids in anions provides evidence for the relative positions of the strain MCS 33T should give us a much broader insight into two acyl functions. By analogy to phospholipids, the loss of the role of polar lipids beyond their involvement in the cell the sn-2-acyl position may be favoured, thus yielding a membrane. Results of such studies may reveal mechanisms more abundant carboxylate anion (Murphy & Harrison, that are valid for many eubacterial cells. 1994). The same seemed to be true here. MS data led to the The ecological niche of strain MCS 33T is of note. The deep identification of the main component as 1-octadecenoyl-2- sea is influenced by three parameters: low temperature, octadecanoyl-3-[O-a-D-glucopyranuronosyl]glycerol-69-N- high hydrostatic pressure and low nutrient concentration. glycine and the minor compound as 1,2-di-octadecenoyl- This last feature is the consequence of the total darkness of 3-[O-a-D-glucopyranuronosyl]glycerol-69-N-glycine. the deep sea, preventing any photosynthesis. Consequently, For phenotypic characterization strains were grown in deep-sea bacteria depend on sinking of material that has 20 ml PYEM medium (2 g peptone, 2 g yeast extract, 0.5 g escaped degradation in the upper layers of the ocean ammonium chloride, 1 litre deionized water). After auto- (Jannasch & Taylor, 1984; Witte et al., 2003). This situation claving and cooling to ambient temperature, 5 ml ribo- is dramatically changed around deep-sea hydrothermal flavin (0.2 mg ml21, sterile filtered), 2 ml 50 % glucose vents. Here a productive ecosystem almost independent of (sterile), 1 ml 20 % magnesium sulphate (sterile) and 1 ml solar energy exists, one which uses oxygen only as the 10 % calcium chloride (sterile) were added and amended product of photosynthesis. It is driven by hydrothermal 21 with 0, 5, 10, 20, 30, 40, 60, 80 or 100 g NaCl l .OD600 of fluids resulting from seawater penetrating into the ocean the cell suspension was determined at the beginning of the crust and heated by magma chambers that leach minerals experiment and after 2 days. The differences between these from the rocks. A shell of warm water is found around two measurements were used to determine salt tolerances. these hydrothermal vents, and this cools to 2 uC, the Growth was tested at different temperatures in SPYEM temperature typical for the deep sea, within a few hundred medium with the same OD protocol. In a medium without metres around the vent (Baross et al., 1982). Strain MCS 33T http://ijs.sgmjournals.org 2211 W.-R. Abraham and others was isolated from a water sample taken at 1762 m depth at Cells are Gram-reaction-negative, rod-shaped, fusiform or 47u 579 N 129u 059 W above a black smoker of the Juan de vibriod, and possess a prostheca varying in length Fuca ridge (Yurkov & Beatty, 1998). Although strain MCS depending on environmental conditions, extending from 33T was isolated from seawater sampled near a hydrothermal one pole as a continuation of the long axis of the cell. vent its temperature requirements did not differ significantly Adhesive material is present at the distal end of the from those of other known cauliform bacteria. The species prostheca. Multiplies by binary fission. At the time of represented by this strain probably lives in a narrow zone division one cell possesses a prostheca and the other a around the hydrothermal vent where the temperature just single polar flagellum. Each appendage occurs at the cell fits its requirement, neither too high nor reaching the usual pole opposite the one formed during fission. The 2 uC of the deep sea. Together with the oligotrophic flagellated cell secretes adhesive material at the base of properties of the strain this points to an interesting the flagellum, develops a prostheca at this site and enters specialization of this micro-organism. The lack of phospho- the immotile vegetative phase. Chemo-organotrophic, lipids reduces the essential amount of phosphorus required strict aerobes. Catalase-, alkaline phosphatase- and leucine by this strain (Van Mooy et al. 2009), which can be an arylamidase-positive but oxidase-negative; requires organic additional advantage for these bacteria to occupy a niche in a growth factors not satisfied by mixtures of B vitamins and thin shell around the hydrothermal vents where the amino acids. Requires NaCl for growth. Growth is temperatures are optimal to support their growth. It remains inhibited or cells become deformed in media containing to be determined whether this species can also be found at 1 % (w/v) or more organic material. The genus is other hydrothermal vents and what its specific roles are in characterized by two major fatty acids, C18 : 1v7c and the food web of hydrothermal vent microbial communities. C18 : 0, and the hydroxy fatty acid C12 : 0 3-OH. Polar lipids The low 16S rRNA gene sequence similarity between strain are a-D-glucopyranosyl diacylglycerol and a-D-glucopyr- MCS 33T and its closest neighbours indicated that this anuronosyl diacylglycerol, while sulfoquinovosyl diacyl strain may belong to a new genus. The analysis of the glycerols and phospholipids are absent. Isolated from whole-cell fatty acids of strain MCS 33T supported this seawater. The type species is Glycocaulis abyssi. view. A number of fatty acids, namely iso-C11 : 0 3-OH, iso- C17 : 0, iso-C17 : 1v9c and C18 : 1v9c, characteristic for the Description of Glycocaulis abyssi sp. nov. Maricaulis group, were absent while C12 : 0 3-OH and C12 : 1 3-OH were found which were absent in Maricaulis strains. Glycocaulis abyssi (a.bys9si. L. n. abyssus depth; L. gen. n. abyssi of/from the depth). While C12 : 0 3-OH is the main hydroxylated fatty acid in Woodsholea and Oceanicaulis species, the C12 : 1 3-OH The description is as given for the genus with the following found in strain MCS 33T is absent in these two genera. In T additions. Cells are rod-shaped, 0.3–0.4 by 0.8–1 mm; the terms of its fatty acid profile, strain MCS 33 is closer to prostheca is about 0.05 mm in diameter. Colonies are species of the genus Hyphomonas than to those of the genus circular, convex and colourless. Can store carbon as poly- Maricaulis (Table 1), although cell multiplication of strain b-hydroxybutyric acid. No acid is produced from carbo- MCS 33T is the same found for Maricaulis species. Strain hydrates and nitrate is not reduced. With the API ZYM MCS 33T differed from Maricaulis spp. and Oceanicaulis system, positive for esterase, esterase lipase, valine aryla- spp. by the lack of sulpholipids and phospholipids and midase, trypsin, a-chymotrypsin, acid phosphatase and from Woodsholea spp. by the lack of sulpholipids and taurine lipids (Abraham et al., 1997). Within the naphthol-AS-BI-phosphohydrolase, weakly positive for Hyphomonadaceae, strain MCS 33T has a unique chemo- cystine arylamidase and b-glucosidase, but negative for taxonomic position based on its lack of phospholipids and lipase, a-galactosidase, b-galactosidase, b-glucuronidase, a- sulpholipids. Also of note is the b-glucosidase activity of glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase strain MCS 33T, which has not been found in any of the and a-fucosidase. In the BIOTYP 100 system, utilizes + type strains of Maricaulis, Oceanicaulis and Woodsholea aesculin, myo-inositol, L( )-tartrate and L-tyrosine but species. The assimilation of malate also distinguished strain not the other substrates. Grows on peptone-yeast extract 21 MCS 33T from these genera. The differential phenotypic media with 30 g NaCl l with optimal growth at 20–60 g 21 21 characteristics between strain MCS 33T and Maricaulis and NaCl l . No growth above 100 g NaCl l . Grows fairly Oceanicaulis species are summarized in Table 3. The 16S well between 15 and 35 uC, while the optimal growth rRNA gene sequence, cellular fatty acids, enzyme activity temperature is 30 uC. The optimal pH for growth is around and lipid pattern of this isolate from the deep sea identify it neutrality; pH range for growth is 6.0–8.0. Polar lipids also as a member of a novel species of a new genus, for which include a-D-glucopyranosyl diacylglycerol-69-N-glycine. we propose the name Glycocaulis abyssi gen. nov., sp. nov. The type strain is MCS 33T (5LMG 27140T5CCUG 62981T), which was isolated from water obtained from Description of Glycocaulis gen. nov. 1762 m depth above the hot water plume of a deep-sea Glycocaulis [Gly.co.cau9lis. Gr. adj. glukus sweet (used to hydrothermal (black smoker) vent near Vancouver island, coin the noun glucose); L. masc. n. caulis stalk; N.L. masc. Canada. The DNA G+C content of the type strain is n. Glycocaulis, sweet (sugar) stalk]. 62.8 mol%.

2212 International Journal of Systematic and Evolutionary Microbiology 63 http://ijs.sgmjournals.org

Table 3. Differential phenotypic characteristics among strain MCS 33T and its closest relatives

Strains: 1, MCS 33T;2,Woodsholea maritima CM243T;3,Oceanicaulis alexandrii C116-18T;4,O. stylophorae GIS-W4T;5,Maricaulis salignorans MCS 18T;6,M. washingtonensis MCS 6T. All data from this study except where indicated. +, Positive; 2, negative; ++, strongly positive; +/2, variable; ND, not determined.

Characteristic 1 2 3 4D 56

Growth temperature range (uC) 15–35 10–40 4–35 15–45 10–40 4–30 Salt tolerance range (g l21 ) 20–60 5–100 20–100 0–90 0–80 0–80 Valine arylamidase ++ + +/2 ++ +/2 Cystine arylamidase +/2 ++/2 ++ +/2 Trypsin +++ ++++++++ a-Chymotrypsin +++ ++++++++ Acid phosphatase ++++/2 ++/2 - Naphthol-AS-BI-phosphohydrolase ++ ++ + + + + b-Glucosidase + 22222 N-Acetyl-b-glucosaminidase 2 + 222 2 Oxidase 2 + +++ + Protease 22 2+ 22 Nitrate reduction 22 + 2 ++ Assimilation of: Malate + 22222 Citrate 22 2+ 22 Arabinose 22 2+ 22 Adipate + 22+ 22 Hydroxy-fatty acid(s) C12 : 0 3-OH, C12 : 1 3OH C12 : 0 3-OH C12 : 0 3-OH C12 : 0 3-OH iso-C11 : 0 3-OH iso-C11 : 0 3-OH

Polar lipids* MGD, MGDOx, GGG MGD, MGDOx, SQD, MGD, MGDOx, PG, ND MGD, MGDOx (PG), MGD, MGDOx (PG), abyssi Glycocaulis Taur SQD SQD SQD DNA G+C content (mol%) 62.8 65.2 61.8 61.6 63.3 63.0

*MGD, 1,2-di-O-acyl-3-O-a-D-glucopyranosylglycerol; MGDOx, 1,2-di-O-acyl-3-O-a-D-glucopyranuronosylglycerol; GGG, 1,2-di-O-acyl-3-[O-a-D-glucuronopyranosyl]glycerol-69-N-glycine; PG, phosphatidylglycerol; Taur, 1,2-diacyl-3-a-D-glucuronopyranosyl-sn-glycerol taurinamide; SQD, 1,2-diacyl-3-O-sulfoquinovosylglycerol. T

DData for O. stylophorae GIS-W4 from Chen et al., 2012. nov. sp. nov., gen. 2213 W.-R. Abraham and others

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